The Kelch-like protein 1 modulates P/Q-type calcium current density

Abstract

The actin-binding protein Kelch-like 1 (KLHL1) is a neuronal protein that belongs to the evolutionarily-conserved Kelch protein super-family. The mammalian KLHL1 is brain-specific, cytosolic and can form multimers and bind actin filaments. KLHL1's function is likely that of an actin-organizing protein, possibly modulating neurite outgrowth, the dynamic morphology of dendritic spine heads; or anchoring proteins essential for post-synaptic function, like ion channels. Targeted deletion of the KLHL1 gene in Purkinje neurons results in dendritic deficits in these neurons, abnormal gait, and progressive loss of motor coordination in mice [He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD (2006) Targeted deletion of a single SCA8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26:9975-9982]. Here we tested the hypothesis that KLHL1 may interact and modulate voltage-gated calcium channels by assessing the interaction of the principal subunit of P/Q-type channels, α1A, with KLHL1. Experiments in human embryonic kidney line HEK 293 (HEK) cells and cerebellar primary cultures revealed co-incidence of α1A and KLHL1 immunoreactivity when testing both the endogenous or epitope-tagged versions of the proteins. Similarly, co-immunoprecipitation experiments in HEK cells and brain tissue exposed the presence of KLHL1 in protein samples immunoprecipitated with FLAG-tagged or α1A antibodies. Functional studies of KLHL1 on P/Q-type current properties probed with whole-cell patch clamp revealed a significant increase in mean current density in the presence of KLHL1 (80% increase; from -13.2±2.0 pA/pF to -23.7±4.2 pA/pF, P<0.02), as well as a shift in steady state activation V50 of -5.5 mV (from 12.8±1.8 mV to 7.3±1.0 mV, P<0.02). Our data are consistent with a modulatory effect of KLHL1 on the P/Q-type calcium channel function and suggest a possible novel role for KLHL1 in cellular excitability.

abstract = "The actin-binding protein Kelch-like 1 (KLHL1) is a neuronal protein that belongs to the evolutionarily-conserved Kelch protein super-family. The mammalian KLHL1 is brain-specific, cytosolic and can form multimers and bind actin filaments. KLHL1's function is likely that of an actin-organizing protein, possibly modulating neurite outgrowth, the dynamic morphology of dendritic spine heads; or anchoring proteins essential for post-synaptic function, like ion channels. Targeted deletion of the KLHL1 gene in Purkinje neurons results in dendritic deficits in these neurons, abnormal gait, and progressive loss of motor coordination in mice [He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD (2006) Targeted deletion of a single SCA8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26:9975-9982]. Here we tested the hypothesis that KLHL1 may interact and modulate voltage-gated calcium channels by assessing the interaction of the principal subunit of P/Q-type channels, α1A, with KLHL1. Experiments in human embryonic kidney line HEK 293 (HEK) cells and cerebellar primary cultures revealed co-incidence of α1A and KLHL1 immunoreactivity when testing both the endogenous or epitope-tagged versions of the proteins. Similarly, co-immunoprecipitation experiments in HEK cells and brain tissue exposed the presence of KLHL1 in protein samples immunoprecipitated with FLAG-tagged or α1A antibodies. Functional studies of KLHL1 on P/Q-type current properties probed with whole-cell patch clamp revealed a significant increase in mean current density in the presence of KLHL1 (80{\%} increase; from -13.2±2.0 pA/pF to -23.7±4.2 pA/pF, P<0.02), as well as a shift in steady state activation V50 of -5.5 mV (from 12.8±1.8 mV to 7.3±1.0 mV, P<0.02). Our data are consistent with a modulatory effect of KLHL1 on the P/Q-type calcium channel function and suggest a possible novel role for KLHL1 in cellular excitability.",

N2 - The actin-binding protein Kelch-like 1 (KLHL1) is a neuronal protein that belongs to the evolutionarily-conserved Kelch protein super-family. The mammalian KLHL1 is brain-specific, cytosolic and can form multimers and bind actin filaments. KLHL1's function is likely that of an actin-organizing protein, possibly modulating neurite outgrowth, the dynamic morphology of dendritic spine heads; or anchoring proteins essential for post-synaptic function, like ion channels. Targeted deletion of the KLHL1 gene in Purkinje neurons results in dendritic deficits in these neurons, abnormal gait, and progressive loss of motor coordination in mice [He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD (2006) Targeted deletion of a single SCA8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26:9975-9982]. Here we tested the hypothesis that KLHL1 may interact and modulate voltage-gated calcium channels by assessing the interaction of the principal subunit of P/Q-type channels, α1A, with KLHL1. Experiments in human embryonic kidney line HEK 293 (HEK) cells and cerebellar primary cultures revealed co-incidence of α1A and KLHL1 immunoreactivity when testing both the endogenous or epitope-tagged versions of the proteins. Similarly, co-immunoprecipitation experiments in HEK cells and brain tissue exposed the presence of KLHL1 in protein samples immunoprecipitated with FLAG-tagged or α1A antibodies. Functional studies of KLHL1 on P/Q-type current properties probed with whole-cell patch clamp revealed a significant increase in mean current density in the presence of KLHL1 (80% increase; from -13.2±2.0 pA/pF to -23.7±4.2 pA/pF, P<0.02), as well as a shift in steady state activation V50 of -5.5 mV (from 12.8±1.8 mV to 7.3±1.0 mV, P<0.02). Our data are consistent with a modulatory effect of KLHL1 on the P/Q-type calcium channel function and suggest a possible novel role for KLHL1 in cellular excitability.

AB - The actin-binding protein Kelch-like 1 (KLHL1) is a neuronal protein that belongs to the evolutionarily-conserved Kelch protein super-family. The mammalian KLHL1 is brain-specific, cytosolic and can form multimers and bind actin filaments. KLHL1's function is likely that of an actin-organizing protein, possibly modulating neurite outgrowth, the dynamic morphology of dendritic spine heads; or anchoring proteins essential for post-synaptic function, like ion channels. Targeted deletion of the KLHL1 gene in Purkinje neurons results in dendritic deficits in these neurons, abnormal gait, and progressive loss of motor coordination in mice [He Y, Zu T, Benzow KA, Orr HT, Clark HB, Koob MD (2006) Targeted deletion of a single SCA8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26:9975-9982]. Here we tested the hypothesis that KLHL1 may interact and modulate voltage-gated calcium channels by assessing the interaction of the principal subunit of P/Q-type channels, α1A, with KLHL1. Experiments in human embryonic kidney line HEK 293 (HEK) cells and cerebellar primary cultures revealed co-incidence of α1A and KLHL1 immunoreactivity when testing both the endogenous or epitope-tagged versions of the proteins. Similarly, co-immunoprecipitation experiments in HEK cells and brain tissue exposed the presence of KLHL1 in protein samples immunoprecipitated with FLAG-tagged or α1A antibodies. Functional studies of KLHL1 on P/Q-type current properties probed with whole-cell patch clamp revealed a significant increase in mean current density in the presence of KLHL1 (80% increase; from -13.2±2.0 pA/pF to -23.7±4.2 pA/pF, P<0.02), as well as a shift in steady state activation V50 of -5.5 mV (from 12.8±1.8 mV to 7.3±1.0 mV, P<0.02). Our data are consistent with a modulatory effect of KLHL1 on the P/Q-type calcium channel function and suggest a possible novel role for KLHL1 in cellular excitability.